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Phase I clinical trials involve assessment of toxicity and adverse effects of the malaria vaccine in human volunteers. A safe dosage is confirmed, the affects of the product on the human body is observed and immune response is measured.
Crucell are developing a recombinant malaria vaccine based on their AdVac technology and produced on PER.C6 production technology. The vaccine is made by inserting the gene for the CSP antigen from the P. falciparum parasite into an adenoviral vector, which functions as a 'vehicle' for vaccination delivery. Recombinant adenoviruses are strong inducers of CMI to heterologous antigens, but the ability to enhance immune responses is weakened by vector immunity stimulated following primary immunization. Clinical trials have been carried out using the replication-defective, recombinant human adenovirus serotype 35 and 26 vectors, which express P. falciparum gene encoding the CS surface antigen. Once the vector carrying the malaria gene is inoculated into PER.C6 cells, a large amount of the vector are produced, making commercial-scale manufacturing of the vaccine possible. The resulting product then undergoes extensive purification before use as a vaccine. The efficacy of the Crucell vaccine candidate was tested in New York University's mouse malaria model. The study showed that a single administration of a prototype AdVac vaccine protects mice upon challenge with the mouse specific parasite.
Studies have also shown that Prime boost regimens, in which CSP antigens are delivered by different adenovirus serotypes, seem to evade this restriction and therefore can be a promising vaccine development using this delivery platform. In 2008, pre-clinical studies for the assessment of the humoral and cellular responses to the prime boost of Ad35.CS.01/Ad26.CS.01 were completed. The prime boost regimen was studied in a group of BALB/c mice, which were immunized intramuscularly with Ad35.CS.01 (prime) followed by a boost with Ad26.CS.01. Using ELISPOT assay a considerable amount of IFN-gamma responses were measured in these mice. ELISA was used to analyse the CS antibody responses, which demonstrated that Ad35.CS.01/Ad26.CS.01 (prime boost) regimen stimulated antibody responses that were higher than responses induced by Ad35.CS.01/Ad26 Empty (prime only). Further immunofluorescence assay (IFA) analysis of the antibody responses on sera samples from Ad35.CS.01/Ad26.CS.01 groups of mice were positive, revealing that antibodies induced by the prime boost regimen could bind P. falciparum sporozoites, whereas poole sera from malaria-naive mice showed negative.
In 2003, this vaccine was tested by itself and in combination with GSK's RTS,S malaria vaccine candidate. These studies have shown that Crucell's AdVac vaccine effectively primed and/or boosted malaria specific immune responses. In 2006, Crucell's vaccine entered a Phase I trialÂ in the US In partnership with the NIAID. The investigation is being conducted on two sites, VanderBilt and Stanford University. The first and second cohorts, involving 18 and 17 volunteers respectively, were enrolled efficiently, with no serious adverse events reported so far. In the following diagram the steps used in producing such a vaccine are outlined in a simplified form.
Figure: Malaria Vaccine Production Process. (from Crucell)
PvRII/AS01B malaria vaccine
The International Center for Genetic Engineering and Biotechnology in New Delhi is developing a P. vivax vaccine candidate PvRII (P. vivax Region II), that is based on DBP, the only invasion pathway for this parasite. This protein allows P. vivax to bind to receptors on red blood cells which leads to invasion of these cells. Since the vaccine is designed to prevent invasion of red blood cells, it may be able to prevent disease caused by P. vivax. This vaccine has the potential to be used in combination with candidates that target P. falciparum.
Another P. falciparum vaccine candidate, JAIVAC-1, consists of a combination of the receptor-binding domain, PfF2, of EBA-175 and the conserved C-terminal 19kD region of merozoite surface protein 1 (PfMSP119) formulated with adjuvant Montanide ISA720. Under cGMP, an Indian biotechnology company, Bharat Biotech have manufactured the clinical grade JAIVAC-1. Toxicology studies with JAIVAC-1 have been completed and an application to conduct a Phase I human trial to evaluate safety and immunogenicity is currently being examined.
In 2009 LaTrobe University's MSP2 recombinant protein (expressed in E.coli) combination 1 (combining the two allelic families 3D7 and FC27)/ adjuvant ISA720 (oil in water emulsion) blood-stage vaccine candidate has completed a Phase 1 clinical trial in Australia to evaluate the vaccine's safety and ability to elicit immune responses. MSP2 is a ~28kDa protein that is anchored in the membrane of the merozoite by a C-terminal GPI anchor moiety. Studies have shown that immunization of mice with MSP2 peptides partially protects mice against challenge with virulent rodent parasite, and antibodies to MSP2 have shown to inhibit invasion of merozoite in vitro. A number of sero-epidemiological studies have demonstrated association between antibody responses to MSP2 and resistance to infection or disease.
Combination B is a blood-stage vaccine that targets MSP1 and MSP2. Both antigens are essential for blood stage merozoite invasion of new red blood cells (17). Thus, MSP-1 and MSP-2 antibodies target viable merozoites in the blood, preventing further erythrocytic infection. This vaccine involves the combination of MSP-1 and MSP-2 with P. falciparum ring-stage infected erythrocyte surface antigen (RESA) in an adjuvant formulation. Hence, the aim of the vaccine is to enhance cellular immunity, resulting in the lysis of infected red blood cells.
In a Phase I/IIb trial in Papua New Guinea, MSP2 (the 3D7 allelic family) was a constituent of an Australian Combination B vaccine candidate that showed a 62 % decrease in parasite density in 120 vaccinated 5 to 9 year-old children. Genotype analysis of breakthrough parasites demonstrated an increase in the dimorphic form of MSP-2. A 1 year observation report of the trial cohort later concluded a higher incidence of morbidity associated with P. falciparum infections of the MSP2-FC27 allelic family in the group that received Combination B, demonstrating that activity of the Combination B candidate vaccine seemed to be partially because of the MSP2 (3D7) component of the vaccine. Because malaria infections stimulate antibodies to MSP2, most people living in regions with high transmission of P. falciparum develop increased titers of anti-MSP2 antibodies by the age of five. Currently, a new form of the vaccine is under development which includes both variants of MSP-2 for targeting both genotypes.
Recombinant vaccine based on blood stage antigen SERA
A group in Osaka university are developing a malaria vaccine using SE36, a recombinant protein based on an amino acid sequence present in the serine repeat antigen (SERA) of malaria parasites. Studies have shown that naturally acquired immunity against malaria is completely correlated with the development of anti-SERA IgG3 antibodies and many types of animals, including chimpanzees, develop antibodies upon vaccination with SE36 that inhibit the growth of malaria parasites. Researchers have constructed a system by which the SE36 malaria vaccine can be mass produced. A phase I clinical trial has been completed in Japan with SE36 to assess its safety and immunogenicity. Among vaccine administered volunteers, 100% of sero-conversion has been reported without any serious adverse effects. Further clinical trials are being planned in endemic regions including Africa.
The function of SERA molecule in the parasite and the host immune response against SERA is also being studied and a new research project for P. vivax vaccine development is being conducted by collaborating with foundations in Uganda, Thai, Indonesia and Solomon. to improve efficacy of SE36 vaccines, a vaccine adjuvant and its mechanism(s) of action is being identified, for example functions of antigen presenting cells and its innate and adaptive immune activation cascades.
Figure: Processed fragments of P. falciparumSERA of the recombinant SE36 protein.
Figure: IFA showing that SE47' is associated with the merozoite's surface.
Figure: Immunoelectron microscopic images using immunogold and anti-SE47'antibodies demonstrate that SE47'are localized in the parasitophorous vacuole around the merozoites. (from Osaka University)
Researchers are investigating different apical membrane antigen 1 (AMA1) vaccine formulations. AMA1 is a protein found at the apex of the blood stage (merozoite) of the malaria parasite. This vaccine would induce production of AMA1 antibodies that would restrict merozoites from infecting and damaging red blood cells.
In order to achieve higher and longer-lasting antibody responses, AMA1-C1 (AMA1 recombinant protein, combination 1) formulated in water-in-oil adjuvant, Montanide ISA 720, is being evaluated in Phase I trials. Compared to AMA1-C1/Alhydrogel, AMA1-C1/ISA 720 has been shown to increase and prolong the specific antibody response in animal studies.
Researchers are also developing AMA1 (of the 3D7 allele) with GSK adjuvants and AMA1-C1 (mixture of 3D7 and FVO) with alum, CpG. Alhydrogel-based vaccines can be improved wth the addition of a second adjuvant, CPG 7909. The combination of adjuvants, studies in animal models have shown that Alhydrogel+CPG 7909 has induced increased levels of antibody production to date for AMA1, and the antibody levels correlated with the inhibition of parasite growth in vitro.
No significant safety issues has been identified so far in PhaseÂ I clinical trials of AMA1 formulated with Alhydrogel+CPG 7909 in U.S, but there are some concerns regarding the induction of autoimmune disease with the use of this DNA analogue. As clinical evaluation is progressing into the field, researchers are careful to monitor specific laboratory markers of autoimmune disease (anti-dsDNA and urine for blood and protein) and clinical evidence of autoimmunity.
BSAM-1 based malaria vaccine
Currently development of a tetravalent vaccine, called BSAM-1, is undergoing clinical trials. The vaccine contains two polymorphic forms for two antigens, AMA1 and MSP1(42). For blood-stage vaccines, MSP1(42) is an alternative major antigen candidate. The vaccine also includes several distinct blood-stage parasite proteinsÂ toÂ increase the possibility of the vaccinee producing sufficient immune response, and the consequent immune response may be additive or synergistic as a result of targeting two different proteins at the same time.
Pre-clinical studies in mice, rats, and rabbits of BSAM-1/Alhydrogel and BSAM-1/Alhydrogel+CPG 7909 demonstrated that these formulations are safe and immunogenic. When the immunogenicity of the BSAM-1 formulations was compared to the immunogenicity of the individual components (AMA1-C1 or MSP1(42)-C1) made at similar dose and formulation, the BSAM-1/Alhydrogel formulations no showed immunologic inhibition. Immune responses induced by anti-AMA1 or anti-MSP1(42) antibody in response to BSAM-1 vaccination were similar to those produced by vaccination with either AMA1-C1/Alhydrogel or MSP1(42)-C1/Alhydrogel. After the addition of CPG 7909 to BSAM-1 formulation, a major increase in the overall production of antibody could be detected.
MSP1 based malaria vaccine
MSP1 is synthesized as a ~200 kilodalton (kDa) polypeptide and is processed, at or before merozoite release from the red blood cell, into smaller fragments that form a noncovalently associated complex. The 42 kDa C-terminal fragment of MSP1 (MSP1(42)) tethers the complex to the surface of the merozoite. During merozoite invasion of red blood cells, secondary processing of MSP1(42) is important for invading parasites. MSP1(42) molecule can be separated into conserved and dimorphic regions.
MSP1(42) antigens have been formulated with Alhydrogel and Alhydrogel+CPG 7909 for clinical testing. Almost no strain specificity in the antibody response to the dimorphic forms of this protein could be observed in human subjects. However, specific T-cell response analysis have demonstrated the significance of the dimorphic region. When compared to Alhydrogel alone, considerable increase in antibody responses was observed when MSP1(42) was formulated with Alhydrogel+CPG 7909. But only depleted parasite growth inhibition was detected in vitro, and inhibition may require much higher antibody levels.
Present clinical studies are focusing on either MSP(42) or a more processed form of the 19 kD fragment. These were expressed by itself or as part of fusion antigens in E. coli, baculovirus or yeast. Studies have shown that MSP-1 42 kD and 19 kD recombinant proteins can protect both Aotus monkeys and mice against fatal parasite challenge. However, a Baylor University (USA) group conducted a Phase I trial of the 19 kD fragment which established that the vaccine had poor immunogenicity and intolerable side effects.
Other antigen based vaccines: A LSP vaccine based on glutamate-rich protein (GLURP) and MSP3 are both undergoing clinical studies. Phase Ia clinical trial of GLURP formulated in Montanide ISA 720 and alum has been carried out.
Additional two antigens, erythrocyte-binding antigen (EBA-175) and its paralog in P. vivax, the Duffy binding protein (PvDBP), are being developed as recombinant vaccine candidates which are expressed in either Pichia pastoris or E. coli or as a DNA prime-boost vaccine. In US, a Phase 1 clinical trial of an EBA-175 candidate has already taken place.
P. vivax is less deadly than P.falciparum, but it is a major cause of malaria in Asia and South America.
These vaccines aim to induce ookinete-blocking antibodies against sexual stage or mosquito antigens for preventing development of infectious sporozoites in the mosquito's salivary glands. These candidate vaccines contain either the P. falciparum ookinete surface protein antigens Pfs25 and Pfs28 or their P. vivax homologues, Pvs25 and Pvs28. These are essential for ookinetes to penetrate the mosquito's gut, where they can develop into the subsequent oocyst stage. Human antibodies induce an immune response against these antigens, which inhibit development of Pfs25 and Pfs28-expressing ookinetes, thus preventing further development and transmission to humans. In a clinical trial the protein Pvs 25 from P. Vivax was used as vaccine, where immunized individuals produced antisera which was present in the mosquito's blood meal. The mosquito that ingested such a blood meal prevented it from transmitting the parasite to any further victim. A recombinant vaccine was produced in yeast (S. cerevisiae) that went through a phase I clinical trial and no serious adverse effects were reported. These proteins are not expressed in the human host and are may not be subject to naturally occurring immune selection pressure. Initial human Phase I trials of Pfs25 and Pvs25 formulated in both ISA51 adjuvant and alum have been performed.
Phase II clinical trials involve assessment of the levels of protection. Safety and potential side effects are monitored by measuring immune response, preliminary efficacy against infection, and determining optimum dosage and schedule.
In Phase 2a trials, malaria-naÃ¯ve volunteers in non-endemic countries are vaccinated and later exposed to malaria-carrying mosquitoes to see how long it takes them to become infected. At the first sign of infection, volunteers are treated with a malaria drug. Phase 2a trials give initial indication of a vaccine's efficacy before the vaccine moves to Phase 2b endemic-country trials. If the vaccine performs well in a series of Phase 2 trials, it moves to Phase 3 trial.
PRE-ERYTHROCYTIC STAGE VACCINES
Whole sporozoite-based vaccines (Sanaria, PfSPZ)
Sanaria, Inc.'s pre-erythrocytic stage malaria vaccine candidate, PfSPZ (P. falciparum sporozoites), uses whole parasites approach to prevent infection. Live, attenuated parasites in the form of sporozoites are extracted from the salivary glands of P. falciparum-infected irradiated mosquitoes, purified, and used for vaccine formulation. Studies have shown radiation-attenuated (weakened) sporozoites to confer high levels of protective efficacy from experimental challenge in humans, when administered as ~1,000 immunizing mosquito bites. In 2009, a Phase I/IIa clinical study was initiated among US volunteers to determine the protective efficacy of whole radiation-attenuated sporozoites, delivered for the first time by needle and syringe. Currently, the attenuated sporozoites can only be stored in cryopreservation conditions and must be injected intravenously. The cost of production might be very high and systems allowing field delivery are not yet clear.
Second Military Medical University in Shanghai developed a MSP1/AMA1 fusion antigen using a yeast expression system (Pichia pastoris), and the use of Montanide ISA 720 adjuvant demonstrated good immunogenic response in rabbits and non-human primates. WHO, MVI and Sinbiomed Inc. collaborated with this University to conduct phase I evaluation on this recombinant vaccine candidate (PfCP 2.9) which showed strong abilty to generate an immune response with only mild adverse effects. In 2008, Sinobiomed was approved to undertake Phase II clinical trial of PfCP2.9 vaccine in endemic areas.
LSA1 based malaria vaccine
LSA1 is a protein expressed on pre-erythrocytic P. falciparum liver stage parasites and is synthesized after the invasion of hepatocytes by sporozoites. LSA1 production continues to increase as the liver cycle. T-cell responses to LSA1 have been associated with protection in individuals naturally exposed to malaria and also those immunized with irradiated sporozoites. However, LSA1 function is still not known. A phase 1/2a clinical trial has been carried out in which LSA1 was injected into two groups of volunteers, where each group received a different adjuvant with the injection. The vaccine showed no efficacy and no serious adverse side-effects were reported. This demonstrates that the vaccine showed no delay in the initiation of infection and protection against the infection was not observed.
CSP-HBc particle vaccine, ICC-1132
Another approach used to develop synthetic vaccines is the incorporation of numerous copies of a CSP epitope into a multiple antigenic peptide (MAP). An immune response was mediated by the vaccine in only those human subjects with cognate HLA haplotypes. In order to evade MHC restriction, the peptide was coupled to a "universal" T-cell epitope, and this construct induced a strong immune response in individuals with different genetic background. From these MAP trials, B-cell and T-cell epitopes have been included into a recombinant Virus Like Particle (VLP) which is based on hepatitis B core (HBc) particles. In US, this CSP-HBc particle vaccine, called ICC-1132, is currently being developed. However, so far the results obtained from Phase II trials have been very poor.
DNA vaccine, MuStDO-5
A pre-erythrocytic stage multiple antigen DNA vaccine, known as MuStDO-5, are being developed using viral vector delivery systems to encode five separate liver-stage antigens: LSA1 and LSA3, CSP, exported protein 1 (EXP1) and sporozoite surface protein 2 (SSP2, also called thrombospondin-related adhesive protein, TRAP). In studies in endemic areas, LSA-1 and LSA-3 have been with protective immunity. The MuStDo-5 vaccine is produced as a combination of five different plasmids. After administering it with GM-CSF DNA as an adjuvant, the vaccine remained harmless and tolerable in mice and rabbits, but demonstrated weak immunogenicity in primates. These studies were conducted in support of a phase I/II clinical trial in healthy volunteers in which the MuStDO 5 pDNA vaccine was monitored for safety, immunogenicity, and protection from challenge with live P. falciparum. Competition between the plasmids was also observed because of the immunodominance of one of the antigens.
Pre-erythrocytic and blood stage viral vector vaccines
In Oxford, UK, a phase I/ IIa clinical trial of two vaccines, FP9/MVA ME-TRAP and PEV3A, was performed on healthy local volunteers, which are active against parasites in pre-erythrocytic liver and blood stages. PEV3A includes peptides from both the pre-eythrocyte CSP and the blood stage antigen AMA1, delivered with influenza virosome. These are virus-like particles that have similar cell binding and membrane fusion properties of the native virus, but lack the viral genetic material. PEV3A and AMA1 were chemically coupled to the virosome's surface to increase their immune response.
Oxford University developed a FP9/MVA ME-TRAP which is a fowlpox strain FP9 and modified vaccinia virus Ankar (MVA) vector which expresses the pre-erthyrocyte antigen TRAP fused to a multi-epitope (ME) string. This viral vector vaccine has demonstrated the association between the stimulation of IFN-gamma producing CD4+ and CD8+ T cell responses and safety against malaria in mice.
Phase I/II studies have been carried out in human volunteers in Gambia, where the subjects were vaccinated with PEV3A alone or in combination with FP9/MVA ME-TRAP. PEV3A vaccinated subjects induced specific immune responses, but no subjects were entirely protected from malaria. PEV3A stimulated an increase in antibody titres and these antibodies bound parasites in immunofluorescence assays; and sporozoite challenge boosted the vaccine-induced immune response. In Kenya the FP9/MVA ME-TRAP prime-boost immunization regimen evaluation showed considerably low immune response and no efficacy. A number of the individuals experienced local pain and other common symptoms.
Pre-erythrocytic stage viral vector vaccines
Researchers are also using simian adenovirus strain 63 (AdCh63) to express ME-TRAP. The AdCh63 ME-TRAP/MVA ME-TRAP prime boost combinations are undergoing clinical trial evaluations with potential dual antibody and cellular immunogenicity detected in a Phase I trial in 2008. Usually researchers prefer to develop protein vaccines that do not contain viral or other recombinant nucleic acid sequences due to the low risk involved from recombination and horizontal gene transfer that may form more fatal parasites in addition to novel bacterial and viral pathogens.
BLOOD STAGE VACCINES:
WRAIR AMA1 (3D7)/GSK AS01B/AS02A
The development of AMA1(of the 3D7 allele) with GSK adjuvants are under clinical trials. A Phase I double blind randomized controlled trial was conducted in Mali, West Africa. The malaria vaccine FMP2.1/AS02A is a recombinant protein (FMP2.1) based on AMA1 from the 3D7 clone of P. falciparum, formulated in the Adjuvant System AS02. Three doses of vaccine were given to 100 children at 0, 1 and 2 months, and children were followed for 1 year. Levels of anti-AMA1 antibodies measured by ELISA increased significantly in all 3 malaria vaccine groups, and remained high during the year of follow up. The FMP2.1/AS02 vaccine had a good safety profile, was well-tolerated and induced high and sustained antibody levels in malaria-exposed children. A Phase I study of the vaccine took place in US and at present it is being evaluated in a Phase II efficacy trial in children at this site in Mali.
A phase II trial of a bi-allele combination AMA1 vaccine (AMA1-C1) formulated with Alhydrogel has been tested in Malian children. This AMA1 vaccine (AMA1-C1) is the combination of two different polymorphic forms of the antigen formulated with a single adjuvant. Alhydrogel is an aluminum hydroxide gel which was selected as the main vaccine adjuvant platform because ofÂ its safety report when used with other recombinant proteins in many age groups and populations. In animal studies alhydrogel formulations of blood-stage vaccine candidates have produced a significant immune response and are safe. So far, there have been four human clinical trials using the recombinant AMA1-C1/Alhydrogel vaccine. Results obtained from a PhaseÂ I trial in malaria-naÃ¯ve U.S. adults were enough to proceed to safety evaluations in semi-immune adults in Mali. This formulation has advanced to phase II trials in Malian children with sufficient safety but with low immunogenicity and no clinical efficacy. In addition, a US PhaseÂ I trial was also conducted to observe the B-cell and T-cell immune responses to vaccination with this formulation.
Other AMA1 vaccine candidates: In 2009, the Biomedical Promate Research Centre in Netherlands carried out a Phase IIa trial of an AMA1 vaccine, which is expressed in the yeast, Pichia pastoris, and formulated with 3 adjuvants. Using engineered fusion proteins, the vaccine development is progressing towards a effective strategy which includes 3 alleles of AMA1 in combination with MSP1(19).
Studies in human subjects in Kenya, Mali and the USA have shown that MSP1(42) antigen fragment formulated in AS02 adjuvant have induced adequate safety and effective immunogenicity. However, in a Phase II trial in Kenya the vaccine demonstrated no efficacy.
Pasteur Institute/African Malaria Network Trust (AMANET) malaria vaccine candidates
Pasteur Institute developed the MSP-3 vaccine candidate as a long synthetic peptide (LSP), as well as a recombinant protein. The MSP3 LSP vaccine construct consists of B and T-cell epitopes that were chosen because of their ability to target via cytophilic antibodies that act upon the monocytes in the antibody-dependent cellular inhibition (ADCI) assay. AMANET partnered with EMVI to sponsor and organise a recently completed Phase I evaluations of this vaccine in children in both Burkina Faso and Tanzania, where the vaccine showed increased levels of safety. In a mouse model of infectious P. falciparum the vaccine-induced antibodies established ADCI activity both in vitro and in vivo. Phase IIb trial of the MSP3 LSP vaccine candidate is currently underway in Mali.
In sub-Saharan Africa, in Gabon, phase II clinical trials of a MSP3/GLURP fusion protein, called GMZ2, which is expressed in the bacteria, Lactococcus lactis, is in progress. GMZ2 would be a very distinctive malaria vaccine because it is a hybrid molecule with two potential targets on the malaria parasite; GLURP and MSP3.
In Phase III clinical trials the vaccine safety is continued to be monitored, potential side effects and efficacy are also examined. The regulatory approval for distribution is obtained (licensure) and the use of vaccine is initiated (introduction).
GSK RTS,S AS01/AS02
Among all malaria vaccine candidates, RTS,S is the most promising and advanced, This vaccine is not designed for travellers but for children living in malaria-endemic areas. This candidate vaccine is a recombinant protein developed by GSK, which includes antigenic C-terminus (207-395 amino acids) of the CSP from malaria P. falciparum, which fuses with hepatitis B surface (HBs) antigen and can be expressed in the form of virus like particles in the yeast, Saccharomyces cerevisiae. CSP antigen induces antibodies that are able to prevent sporozoites from invading hepatocytes, and a cellular response that is able to remove infected hepatocytes. CSP is fused to HBs antigen to develop into a more potent vaccine. When combined with a GSK adjuvant system, RTS,S stimulates the production of antibodies and white blood cells that reduce the malaria parasite's abilty to infect, survive, and develop within the human liver. Besides stimulating partial protection against malaria, the RTS,S vaccine candidate can also induce a protective immune response to hepatitis B, which is a widespread infection in developing countries.
The first Phase I studies of RTS,S formulated with GSK AS02 adjuvant (which contains an oil-in-water emulsion, MPL and QS21) showed protection against malaria in 6 out of 7 human volunteers. The consequent Phase IIa clinical trials in 100 vaccinated volunteers demonstrated a general protective efficacy of 30-40%. The efficacy against delayed re-challenge seemed to be decreased. Additional trials in Gambian adults showed a 30% protection against malaria infection for more than 15 weeks. However the results obtained from this study indicated a decline in efficacy after 9 weeks. the next year, Before the malaria season began, the volunteers who were immunised with a fourth dose of the vaccine, once more revealed a 47% protection against infection within 9-week observation period. In Mozambique Field testing on 2022 children aged between the age of 1 to 4 years confirmed that three doses of RTS,S/AS02 reduced the first malaria clinical episode rate at efficacy of about 30%, and caused a decrease of 37% in blood parasitemia occurrence within six months, and a ovrall decrase of 57% in severe disease prevelance. This protection lasted for 18 months. The current limitation is that the Follow-up report of long-lasting immunity and protection seems to rapidly decline within 18 months; however, these estimates may be inaccurate due to the decrease in the levels of disease in the older age groups. Another study of RTS,S/AS01 (using liposomes in the adjuvant preparation) in Tanzania and Kenya of more than 800 infants aged 5 to 17 months demonstrated efficacy of 55% against all clinical malaria events over 8 months of follow-up. Yet again severe malaria efficacy revealed only 1 event in the vaccine group and 7 events in the control gruops. A long-term follow-up report is required for this study.
In May 2009 the most crucial multi-country Phase III registration trial of RTS,S/AS01 began and will include 11 sites in Mozambique, Gabon, Kenya, Tanzania, Malawi, Burkina Faso and Ghana. The Phase III trial will evaluate the vaccine's efficacy in two groups of children. One group is infants aged 6 to 12 weeks and the second group includes children aged 5 to 17 months. From these studies an appropriate dosage schedule will be characterised for the RTS,S vaccine, best adjuvant will be evaluated, and RTS,S will be administered with other vaccines hoping to confer longer-lasting protective immunity. Despite slow progress, researchers are optimistic that between 2014 and 2015, the licensure and introduction of the first malaria vaccine could take place.
Unsuccessful Phase III vaccine trials:
The SP66 vaccine (also known as Patorraya or Cocktail vaccine) is the first vaccine developed in 1987 and involves a combination of antigens from the sporozoite (CS repeats) and merozoite parasites. This vaccine was developed in Colombia as a synthetic multi-epitope, multistage peptide vaccine which is formulated with alum as an adjuvant. Phase I trials of the SPf66 vaccine candidate showed 75% efficacy rate and was immunogenic and well tolerated by volunteers. SPf66 consists of 3 peptide epitopes from 3 blood stage proteins (35 KD, 55KD and 83 KD) intercalated with NANP sequence. The aim of this vaccine is to inhibit the parasite at its later merozoite form, as it appears from initial incubation in liver. The vaccine induces antibody production which would prevent the parasite from infecting red blood cells.
Phase IIb and III trials in both low and high malaria endemicity areas in Latin America and Africa were disappointing, as the study showed decreased efficacy of 38.8% and 60.2%. A trial carried out in Tanzania showed the efficacy to be 31% in children (1 to 5 years old) after a year of follow-up. However another study in Gambia in infants (aged 6-11months old) did not show any effect in spite of the lengthy trial periods and the number of studies conducted. Several Phase III field trials of the vaccine were later tested in thousands of volunteers, but the efficacy was established to be too low and inconsistent to authorise further development of the vaccine. The process by which the vaccine induces immunity is still unknown, and thus it has less likelihood of preventing malaria.
The highly attenuated NYVAC vaccinia virus strain is a multistage vaccine that has been utilized to develop a multiantigen candidate for malaria. Seven P. falciparum antigenic genes derived from the sporozoite phase (CSP and sporozoite surface protein 2 (called PfSSP2). LSA1, three antigens from the blood stage (MSP1, SERA and AMA1) and one sexual stage antigen (Pfs25) were inserted into a single NYVAC genome to generate NYVAC-Pf7. All seven antigens were expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was established. Initial preclinical trials in Rhesus monkeys showed promising results where 4 out of the 7 antigens produced specific antibody responses (CSP, PfSSP2, MSP1 and PFs25) and thus NYVAC-Pf7 was safe and well tolerated. Clinical trials in humans showed effective immunogenicity in more than 90% of volunteers, but they had very low antibody responses. However, administration of the vaccine when these volunteers were challenged with P.falciparum, some subjects had complete protection. This study has led to warrant of ongoing trials.
Stage of Plasmodium
Irradiated sporozoites, CSP or peptides; LSA-1
Merozoite and Erythrocytes
EBA-175; MSA-1,2,3,4 and 5; RESA; SERA; Rhoptry Associated Protein (RAP); Histidine Rich Protein (HRP); AMA1and GLURP.
Pfs 25, Pfs 48/45, Pfs 230
SPf 66 (pre-erythrocytic and asexual blood stageproteins of P.falciparum)